The present invention relates to a method for isolating nucleic acid from a sample.
Recent developments in molecular biology such as, nucleic acid amplification methods, recombinant DNA techniques and sequencing methods rely on isolated nucleic acids as input material. Thus there is an ongoing need to improve the methods presently used for the purification of nucleic acid from complex samples. Of course these methods should be as simple and fast as possible and should result in a high recovery of the nucleic acid from the starting material. Furthermore it would be desirable to have methods that can be readily automated. Starting materials from which DNA is to be isolated are usually complex biological materials in which the nucleic acid is present surrounded by cellular material such as proteins or lipids. Such materials may be, for example, whole blood, blood serum, buffy coat, urine, faeces, liquor cerebrospinalis, sperm, saliva, cell cultures etc.
Known methods for the isolation of nucleic acid from these materials may comprise the use of a chaotropic substance, for example to lyse cells present in the starting material, and free the nucleic acid therefrom. By chaotropic substance is meant any substance capable of altering the secondary, tertiary and/or quatemary structure of proteins and nucleic acids, but leaving at least the primary structure intact. Examples of chaotropic substances are guanidinium (iso) thiocyanate and guanidine hydrochloride. Also sodium iodine, potassium iodine, sodium (iso)thiocyanate, urea or mutual combinations therewith are suitable.
When a nucleic acid containing material is treated with a chaotrope and contacted with a nucleic acid binding solid phase, the nucleic acid will bind to the solid phase in the presence of the chaotrope and the solid phase with the nucleic acid bound thereto can thus be separated from the remainder of the sample. Solid phases used in these processes usually comprise siliceous material such as glass particles, either porous or non-porous, silica gel, glass fiber, filters made of siliceous material, diatomeceous earth, etc. Such a method, based on the use of a chaotropic substance and a nucleic acid binding solid phase is described in EP389063, the contents of which are herewith incorporated by reference. In this patent a method for the isolation of nucleic acid is described based on the use of a chaotropic substance and silica or a derivative thereof. The method can be used for the isolation of nucleic acid from complex biological starting material. With the method as described in EP 389063 nucleic acid is bound to silica in the presence of a chaotrope. Thereafter the solid phase with the nucleic acid bound thereto is separated from the liquid and washed.
In practice, washing is usually performed in a sequence of washing steps, The solid phase is usually washed with a high salt buffer, that resembles the binding buffer in its constitution. One or more wash step with a lower alkyl alcohol, such as ethanol 70% are part of the washing procedure, to remove proteins, lipids and the like that may also have been bound to the silica. The washing procedure may also involve an additional wash step with acetone to remove any remaining impurities from the silica. The volatile acetone is removed again by drying the solid phase prior to the elution of the nucleic acid therefrom with a low salt buffer.
Prior art procedures have the disadvantage that they are still rather time consuming due to these complicated washing procedures involved. Especially the use of organic solvents like acetone also makes it more difficult to automate the process. The volatile nature of the solvents used puts severe constraints on a possible apparatus to be designed for performing these methods in an automated manner.
Thus a need exists for methods that are less complicated as far as the washing procedure is concerned and thus easier to perform in an automated manner. The present invention provides such a method. The present invention resembles prior art methods in that it is based on the binding of nucleic acid to a solid phase in the presence of a chaotropic substance. However, with the method of the present invention the washing procedure has been simplified and the need for washing with acetone or drying the solid phase prior to elution is eliminated.
The method of the present invention for the isolation of nucleic acid from nucleic acid containing starting material is a method wherein the starting material is contacted with a chaotropic substance and, either simultaneously or subsequently, with a nucleic acid binding solid phase, where after the nucleic acid binding solid phase is subjected to a washing procedure and, optionally, the nucleic acid is eluted from the nucleic acid binding solid phase, characterized in that the washing procedure comprises the steps of washing the solid phase with subsequently
one or more high salt buffer solution(s)
optionally an alcoholic solution
a low salt buffer solution.
In nucleic acid isolation methods guanidine(iso)thiocyanate is often used in lysis buffers.
However, in cases where the nucleic acid to be isolated is intended to be used as input material for amplification reactions the guanidine ions may disturb the amplification reaction if they are not washed away properly. The high salt buffer used in the washing procedure of the method of the present invention preferably is NaSCN. By washing with NaSCN the guanidine ions that were present in the lysis buffer are replaced by natrium (sodium) ions of the high salt wash buffer.
With the method of the invention(one on the high salt buffer(s) used may comprise 1-10 M NaSCN/10 mM tris. The pH of such buffers may vary from 6 to 8 with an optimum around 6.5. Good results were obtained with a buffer having a NaSCN concentration of 10 2-5M/10 mM tris and a pH of 6.5.
Another high salt buffer that may be used in the washing procedure that is part of the method of the present invention may comprise NaCl preferably in a concentration between 1-10M. A preferred NaCl buffer comprises 2-5M NaCl/10 mM tris and has a pH of about 6.5.
To facilitate the transfer from chaotropic high salt buffer to the low salt buffer used in the washing procedure, it is advantageous to wash the solid phase with the nucleic acid bound thereto first with a NaSCN containing buffer (which will result in the replacement of guanidine ions by natrium ions) and subsequently with a NaCl buffer (resulting in the replacement of thiocyanate with chloride ions).
Thus the washing procedure may comprise the subsequent steps of,
washing with a first high salt buffer comprising 1-10M NaSCN/10 mM tris, with a pH approximately between 6 and 8 and
washing with a second high salt buffer comprising 1-10M NaCl/10 mM tris, with a pH approximately between 6 and 8, with a preference for a concentration of 2-5M of NaSCN and NaCl respectively and a pH of 6.5 for both buffers.
The wash steps with the high salt buffers may be followed by washing with a lower alkyl alcohol, for example, ethanol or isopropanol, to decrease the concentration of the salts (i.e. NaSCN and NaCl) from the solid phase in the sample. Furthermore, the alcohol wash steps decrease the concentration of lipids in the sample. In the case ethanol is used the preferred concentration range is about 60-70% ethanol. A most preferred concentration of ethanol is 63%. In the case of isopropanol an concentration of about 50% is preferred. The method may comprise more than one wash step with the alcoholic solution. Good results were obtained with a method comprising two subsequent washes with 70% ethanol.
The washing procedure of the method according to the present invention involves washing with a low salt buffer solution after washing with (one or more) high salt buffer(s) and optionally with a lower alkyl alcohol. With the use of the low salt buffer ethanol is removed from the solid phase. The use of the low salt buffer eliminates the need for an acetone wash and subsequent drying prior to elution of the nucleic acid from the solid phase. Elution normally is carried out with a low salt buffer or water. It is therefore surprising that the washing of the solid phase with a low salt buffer solution as performed with the method according to the invention, does not result in elution of a substantial part of the nucleic acid from the solid phase.
A preferred low salt buffer used with the method of the invention comprises 2-30 mM NaCl/10 mM tris. Good results were obtained with a low salt buffer comprising 15 mM NaCl/10 mM tris and having a pH of 6-8.
After washing with the low salt buffer, the nucleic acid may directly be eluted from the solid phase. It has been found that the addition of a weak chaotrope, for example betaine, to the elution buffer has an advantageous effect on the elution. When betaine is used in the elution buffer, the concentration may be around 3 M. Stronger chaotropes may be used in the elution buffer as well, be it in lower concentrations. Any chaotropic agent that is used to enhance the elution of the nucleic acid from the solid phase must be added at concentration that does not disturb any downstream use of the nucleic acid, for instance in nucleic acid amplification methods or direct hybridization methods.